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CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY,1071-412X/98/$04.000

Sept. 1998, p. 609612 Vol. 5, No. 5

Copyright 1998, American Society for Microbiology. All Rights Reserved.

Quantifying Serum Antiplague Antibody with aFiber-Optic Biosensor

GEORGE P. ANDERSON,1* KEELEY D. KING,2 LYNN K. CAO,2 MEAGAN JACOBY,1

FRANCES S. LIGLER,1 AND JOHN EZZELL3

Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Washington, D.C.,1 andGeo-Centers Inc., Fort Washington,2 and Applied Research Division, U.S. Army Medical Research

Institute of Infectious Disease, Fort Detrick,3 Maryland

Received 29 January 1998/Returned for modification 9 April 1998/Accepted 20 May 1998

The fiber-optic biosensor, originally developed to detect hazardous biological agents such as protein toxinsor bacterial cells, has been utilized to quantify the concentration of serum antiplague antibodies. Thisbiosensor has been used to detect and quantify the plague fraction 1 antigen in serum, plasma, and whole-bloodsamples, but its ability to quantify serum antibodies has not been demonstrated. By using a competitive assay,the concentration of serum antiplague antibodies was ascertained in the range of 2 to 15 g/ml. By makingsimple dilutions, concentrations for 11 serum samples whose antiplague antibody concentrations were un-known were determined and were found to be in good agreement with enzyme-linked immunosorbent assay

results. The competitive assay method could be used to effectively determine the exposure to plague of animalsor humans or could be applied to other diseases, such as hepatitis or AIDS, where the presence of antibodiesis used to diagnose infection.

Yersinia pestis, the etiologic agent of bubonic plague, contin-ues to be endemic in many parts of the world. Although itoccurs most frequently as an infection in wild rodents, plaguecan be transmitted to domesticated cats or dogs either by thebites of fleas or through the consumption of plague-infectedcarrion (8, 10). Subsequently, these animals may become vec-tors for transmission to humans. Serologic surveys of wild anddomesticated animals in high-risk areas would permit an earlywarning of the spreading disease (18). The availability of a sim-

ple test performable in the field would allow the rapid screen-ing of animals and people to monitor for exposure or immu-nization effectiveness (15).

Since plague-infected animals produce a strong humoralresponse to the fraction 1 (F1) antigen, the detection of F1antibodies is the basis for standard serological tests in plaguesurveillance and diagnosis (20). The F1 antigen is a protein-polysaccharide complex that forms a major component of theouter membrane capsule of Y. pestis (5). Capsule productionoccurs at 37C upon transmission from fleas to warmer mam-malian hosts. The F1 antigen is thought to confer resistance tophagocytosis (5). The F1 antigen is also thought to be theprimary immunogen in the whole-cell vaccines with protection-inducing properties (19).

The fiber-optic biosensor is being developed to conduct fluo-roimmunoassays in a rapid, user-friendly form (11). The assaysthat have been developed have primarily been for hazardousbiological substances. For example, sandwich immunoassayshave been developed for plague F1 antigen (6), staphylococcalenterotoxin B (17), and ricin (13). Another use for the sensorhas been the detection of small molecules. A competitive assayhas been used to quantify trinitrotoluene contamination ingroundwater (16). The principal advantage of this biosensor isthat it permits samples to be tested in the field. While thestandard enzyme-linked immunosorbent assay (ELISA) takes

a skilled technician in a laboratory several hours to complete,the biosensor is capable of producing an answer within 10 to 20min. In addition, the biosensor has recently been miniaturized(11), and an automated version which will further facilitatesample analysis is in development.

The sandwich fluoroimmunoassay has been the method ofchoice to detect biological molecules with the fiber-optic bio-sensor. In this assay, antibodies directed towards an antigen ofinterest are immobilized on the probe. When the probes are

exposed to an antigen-containing sample, the antigen is boundby the antibody on the probe surface. The amount bound isdetermined by the application of a high concentration of fluo-rescently labeled antibody, which forms a fluorescent complexat the probe surface. The amount of fluorescent complex isquantified by the optoelectronics, which launches excitationlight into the proximal end of the probe and measures the gen-erated fluorescence returning back up the probe (8). Thoughthis method has worked well for toxins and for proteins such asthe F1 antigen, a different assay method was required to quan-tify serum antiplague antibodies.

Several different methods were explored in order to obtainthe most effective protocol for quantifying antiplague antibod-ies (Fig. 1). The first method tested was a competitive assay, in

which probes were prepared by directly immobilizing the F1antigen onto the probe surface. When these probes were ex-posed to serum containing anti-F1 antigen antibodies, a por-tion of the F1 antigen on the probes was bound. This resultedin a decrease of signal generated by the subsequent incubation with a standard quantity of fluorescently labeled antiplagueantibody. The inhibition of signal compared to that of unex-posed probes was indicative of the amount of antiplague anti-bodies in the serum.

A modified competitive assay was also investigated. In thisprotocol, antiplague immunoglobulin G (IgG)-coated probes were first exposed to a limited amount of F1 antigen. Next,they were exposed to the serum sample and finally to thefluorescently labeled antiplague antibody. Again, the degreesof signal inhibition between probes which had and had notbeen exposed to serum were compared.

* Corresponding author. Mailing address: Center for Bio/MolecularScience and Engineering, Naval Research Laboratory, Code 6910,Washington, DC 20375-5348. Phone: (202) 404-6033. Fax: (202) 404-8897. E-mail: ganderson@cbmse.nrl.navy.mil.

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The final method examined was a sandwich immunoassay.Fiber probes with immobilized antiplague IgG were coatedwith F1 antigen and then incubated with serum samples. Thequantity of antiplague serum antibodies which bound to theprobe surface was then determined with fluorescent rabbitanti-human IgG.

MATERIALS AND METHODS

Reagents. The F1 antigen (3), sera from immunized personnel, rabbit anti-plague IgG purified with protein G, and ascites fluid containing the monoclonalantibody YPF1-6H3-1-1-IgG, henceforth referred to as 6H3-IgG, were providedby the U.S. Army Medical Research Institute of Infectious Disease (USAM-RIID). The 6H3-IgG monoclonal antibody was developed at USAMRIID byinjecting F1 antigen (lot 4, produced by J. E. Williams, Walter Reed ArmyInstitute of Research, Washington, D.C.) into BALB/c mice. The F1 antigenpreparation used in this study was the same as that utilized in the hybridomascreening process. 6H3-IgG was affinity purified on a 3-ml Avidchrom column(Unisyn Technologies, San Diego, Calif.), by eluting the bound antibody with 0.1M sodium acetate (pH 3.0) plus 20% glycerol. The purified antibody was thendialyzed three times against phosphate-buffered saline plus 0.01% sodium azide(PBS).

The anti-F1 antigen titers of the serum samples from immunized personnelused in this study were estimated by using twofold dilutions in a capture ELISA.Negative control serum was obtained from Gibco (Gaithersburg, Md.).

Rhodamine-conjugated rabbit anti-human IgG was purchased from AccurateChemical (Westbury, N.Y.). Human IgG containing antiplague antibody waspurified from a sample from an immunized volunteer on an Avidchrom columnas described above. The quantity of antiplague IgG was determined by themodified competitive method described below. Other fluorescently labeled an-tibodies were prepared by dialysis of 1 mg of each antibody (1 mg/ml) against 50mM borate (pH 9.3) plus 50 mM NaCl, followed by dialysis overnight in the samebuffer containing 0.01 mg of tetramethylrhodamine-5-isothiocyanate, isomer G(TRITC) (Molecular Probes, Eugene, Oreg.), per ml. Free dye was removed bygel filtration on Bio-Gel P-10 (Bio-Rad, Hercules, Calif.) equilibrated with PBS.The dye-to-protein molar ratio for each antibody preparation was determined tobe between 1.0 and 1.5 by the method of Amante et al. (1). Bovine serumalbumin (2 mg/ml) was added to the labeled antibody prior to storage at 4C.

Fiber preparation. Optical probes were prepared from plastic-clad opticalfiber with a 200-m-diameter silica core (Quartz Products, Tuckerton, Del.). Thesensing region was formed by removing 12.5 cm of cladding at the fibers distalend to expose the silica core. Residual cladding from the probe area was re-moved by immersion in concentrated hydrofluoric acid for 1 min. The fibers were

subjected to computer-controlled immersion in hydrofluoric acid to form taperedprobes (2).

Rabbit antiplague IgG or F1 antigen was immobilized onto the tapered coreaccording to the procedure of Bhatia et al. (4, 9). Briefly, clean probes wereincubated for 30 min in 4% thiol-terminal silane in toluene (3-mercaptopropyltrimethoxysilane; Fluka, Hauppauge, N.Y.) and then for 1 h with the heterobi-functional cross-linker N-succinimidyl 4-maleimidobutyrate (Fluka) (2 mM) inethanol. Finally, the probes were incubated with the capture antibody or F1antigen at 0.05 mg/ml in PBS for 2 h. This procedure immobilized the antibodyon the fiber surface at approximately 2 ng/mm2 (4). The fiber probes were placedin storage in PBS at 4C. Prior to testing, the probes were placed into flowchambers as previously described (2). All testing was performed with the labo-ratory breadboard biosensor, which has been previously described (12).

RESULTS

Competitive assay with F1 antigen-coated probes. The firstmethod used to quantify serum antiplague antibodies was a

two-step competitive assay. First, a standard response curve forthe probes coated with F1 antigen was constructed. The proberesponse was first standardized by incubation with 200 ng ofTRITC-labeled 6H3-IgG (T-6H3-IgG)/ml for 5 min. Subse-quently, various known amounts of unlabeled 6H3-IgG (0 to100 g/ml) were incubated on the probes for 10 min. Finally, todetermine the degree of binding inhibition, the probes wereincubated with 1,000 ng of T-6H3-IgG/ml. The additional flu-orescent signal generated after 5 min of incubation with 1,000ng of T-6H3-IgG/ml was divided by the signal generated by theinitial exposure to 200 ng of T-6H3-IgG/ml. A ratio (R) can bedescribed by the following equation: R (signal 2 signal 1)/(signal 1 background). The plot of this ratio versus the con-centration of unlabeled 6H3-IgG is shown in Fig. 2. As expect-ed, the ratio dropped with increasing concentrations of unla-beled antibody. However, since very high levels of antibodywere required to inhibit the response, additional methods wereinvestigated.

Competitive assay with rabbit antiplague IgG-coated probes.Probes coated with rabbit antiplague IgG, the same type usedin the previously developed sandwich immunoassay to quantify

F1 antigen (6), were used to develop an assay to quantify se-rum antiplague antibody. The antibody-coated probes werefirst incubated twice with T-6H3-IgG (5 g/ml) for 5 min eachtime to determine nonspecific background signal due to adsorp-tion of the labeled antibody. The probes were rinsed with PBSplus 0.1% Triton X-100 between each step. After the back-ground signal had been determined, the probes were exposedto F1 antigen (200 ng/ml) for 5 min and then to T-6H3-IgG (5

FIG. 1. Schematic of immunoassay methods. (1) Competitive assay with probes coated with F1 antigen. The fluorescently labeled antibodies are indicated by flags.(2) Competitive assay with probes with antiplague antibody that was immobilized and then coated with F1 antigen. (3) Sandwich immunoassay with fluorescentanti-human antibody to generate the signal.

FIG. 2. Competitive assay standard curve for antiplague antibody withprobes with immobilized F1 antigen. An initial signal (signal 1) was generated onthe optical probes by the addition of T-6H3-IgG (200 ng/ml). The probes werethen exposed to various amounts of unlabeled 6H3-IgG. The amount of bindingwas determined by the reapplication of T-6H3-IgG (1,000 ng/ml), yielding signal2. The signal 2/signal 1 ratio versus 6H3-IgG concentration is shown. The ratiosfor two probes are shown at each concentration.

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g/ml) for 5 min to standardize the probe response (signal 1).Following standardization, the probes were reprimed by expo-sure to 500 ng of F1 antigen/ml. Ultimately, either knownamounts of 6H3-IgG in PBS (to generate a standard curve) orserum samples were incubated on the probes for 5 min. Theamount of blocking that occurred was determined by a finalincubation with T-6H3-IgG (5 g/ml) for 5 min (signal 2). Thesignal ratio was determined as described above and plottedversus the concentration of 6H3-IgG (Fig. 3). The curve was fitby using the hyperbolic second-order-decay equation Y (a

bc)/(c X). The fit to this equation was used to calculate theconcentrations of antibody in serum samples. This competitive

method quantified the antibody concentration in the 2- to 15-g/ml range.

Sandwich immunoassay for antiplague antibodies. The finalmethod investigated was a standard sandwich immunoassay.Again, probes with rabbit antiplague IgG were prepared. Thebackground signal generated by 5 g of TRITC-labeled rabbitanti-human IgG/ml was determined by incubating probes twicewith the fluorophore-labeled antibody for 5 min each time, asdescribed above. These probes were then primed with F1 an-tigen (10 g/ml) for 5 min, treated with various amounts ofhuman antiplague IgG for 5 min, and washed, and then theamount bound was determined by incubation with 5 g ofTRITC-labeled rabbit anti-human IgG/ml for 5 min (signal 1).This response was calibrated by incubating the probes with a

standard concentration of human antiplague IgG (10 g/ml)for 5 min followed by a final incubation with TRITC-labeledrabbit anti-human IgG for 5 min (signal 2). The signal 1/signal2 ratio was plotted versus the known amount of human anti-plague IgG (Fig. 4). This method was not utilized to analyzeany samples with unknown amounts of antiplague antibodydue to problems associated with nonspecific adsorption of nor-mal human IgG.

Evaluation of samples with unknown amounts of antiplagueantibody. USAMRIID provided 11 numbered serum samplesto be quantified. These samples were obtained from humanvolunteers who had been immunized against plague (Table 1).By using the modified competitive method with probes coatedwith rabbit antiplague IgG, the concentration of each samplewas determined. After an initial screen, it was sometimes nec-essary to dilute the strongly positive samples with PBS in order

to obtain a ratio on the sensitive portion of the standard curve.Samples that generated a signal ratio of 1.50 or above wereconsidered to contain no detectable amount of antiplague an-tibody.

DISCUSSION

A number of methods to quantify antiplague antibodies withthe fiber-optic biosensor were examined. The competitive as-say with probes coated with F1 antigen failed to provide ade-quate concentration discrimination. While a large signal de-crease occurred after exposure to 5 g of 6H3-IgG/ml, the F1antigen-coated probes required very high concentrations ofantibody (100 g/ml) to block subsequent signal generation.

To circumvent this difficulty, a competitive assay that used

rabbit antiplague probes was tried. In this assay, a small amountof F1 antigen was bound to these probes. This was done tocalibrate the probes response. The probes were then coatedwith a larger amount of F1 antigen. This second amount couldthen be bound by antiplague IgG in the sample or by thesignal-generating T-6H3-IgG. This method resulted in a re-sponse which could discriminate 0 to 15 g/ml of antiplagueIgG/ml.

A sandwich immunoassay with TRITC-labeled rabbit anti-human IgG as the signal-generating antibody was also investi-gated. Not being a competitive assay, this method was expectedto be significantly more sensitive. In addition, the sandwichassay discriminates between immunoglobulin classes, while thecompetitive assays do not. The standard curve did show a limit

of detection improved to 0.25 g of human antiplague IgG/ml(Fig. 4), but this method was abandoned when it was observedthat the control samples often generated signals due to thenonspecific adsorption of normal human IgG. Carvalho et al.(7), who have also investigated various assay formats forplague antibody detection, did not experience this difficulty,indicating that it may be a surface-specific phenomenon.

Of the various methods investigated, the competitive assaywith probes coated with rabbit antiplague IgG was selected foran additional trial to quantify antiplague IgG in a series ofsamples supplied by USAMRIID (Table 1). The correlationbetween ELISA data and that obtained with the fiber-opticbiosensor was excellent. The biosensor identified three nega-tive serum samples while also correctly identifying positivesamples with low (10 g/ml), medium (10 to 100 g/ml), andhigh (100 g/ml) concentrations of antiplague IgG. To quan-

FIG. 3. Competitive assay standard curve for antiplague antibody withprobes with immobilized rabbit antiplague IgG. To obtain the ratios, an initialsignal (signal 1) was generated on the probes by addition of F1 antigen (200ng/ml) for 5 min and then T-6H3-IgG (5 g/ml) for 5 min. The second signal(signal 2) was obtained by repriming the probes with 500 ng of F1 antigen/ml andincubating them with various amounts of 6H3-IgG for 5 min and then withT-6H3-IgG for 5 min. The signal 2/signal 1 ratio versus 6H3-IgG concentrationis shown. The mean standard error for three probes is shown at each concen-tration.

FIG. 4. Sandwich immunoassay standard curve for serum antiplague anti-body. Probes coated with rabbit antiplague IgG were primed with F1 antigen (10g/ml) and incubated with increasing amounts of purified human antiplague IgG.Finally, the amount of human antiplague IgG bound to the probe was delineatedby the binding of TRITC-labeled rabbit anti-human IgG (5 g/ml) for 5 min. Themean standard error for three probes is shown at each concentration.

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